Modares Journal of Biotechnology

Abstract  L-glutaminases (L-glutamine amidohydrolase, 3.5.1.2) belong to the superfamily of serine-dependent β-lactamases and penicillin-binding proteins. L-glutaminases have received much attention in the last few decades due to their catalytic ability to deaminate glutamine to glutamic acid and ammonia; a property that has made them valuable in different industrial applications, especially in medicine. Research on glutaminase has progressed during the last four decades, though slowly in comparison to other industrially important enzymes. The relatively high cost of glutaminase is one of the major drawbacks hindering its industrial applications. The current production levels of glutaminase are also insufficient for the required clinical trials to facilitate its medical uses and for other applications.The purpose of this study is the solubilized recombinant expression and functional assay of L-glutaminase from Alteribacillus bidgolensis (P4BT) in an E. coli BL21 (DE3) expression system. In this study, the L-glutaminase gene was successfully cloned with the pET30a expression vector in the E. coli BL21 (DE3) expression system. Solubilized expression was achieved with the aid of the pG-KJE8 vector, which contains molecular chaperones. Ultimately, the specific activity of the purified and dialyzed enzyme was assessed at 40°C and pH 8, yielding an enzymatic activity of 0.53 ± 0.01 U/mg at a substrate concentration of 8 mM. The Km value for L-glutaminase was calculated at 3.10 mM, with a Vmax of 0.62 U/mg.
 

Abstract Aim: During the uncontrolled development of cells in the body, a subset of neoplasms or tumors is formed, the abnormal proliferation of these cells leads to the formation of a mass and eventually cancer. This mass can spread throughout the body. Thus, inhibiting the abnormal growth of cancer cells will have a significant effect on preventing the spread of cancerous tumors and improving the disease. Therefore, in the present study, a new sulfonamide derivative was designed and synthesized (HB20) and its anti-cancer effects on human breast cancer cell line (MCF-7) were investigated.
Materials and Methods: For the synthesis of a sulfonamide derivative (HB20), dcriptiazonium salt was first made using a sulfamethoxazole base compound and then combined with a pyrimidine coupling agent. Concentrations of a new synthetic compound (HB20) against Cells (MCF-7) were used. MTT assay was also performed to measure survival and cell proliferation.
Results: The synthesized compound structure was confirmed by spectral analysis, such as FT- IR, and NMR. Also, Survival in MCF-7 cells treated with a synthetic compound (HB20) was significantly reduced compared to the control group (untreated). HB20 inhibits the proliferation of MCF-7 cancer cells with an IC₅₀ value of 75/23 μg/ml.
Conclusion: The new sulfonamide derivative (HB20) has the potential to inhibit proliferation and anti-cancer properties in the cell line (MCF-7).

Abstract Hsp70 family members are central components of the cellular network of molecular chaperones and folding catalysts. The gene encoding a protein related to Hsp70 or DnaK in the domain bacteria is called dnaK. DnaK proteins are involved in de novo protein folding, formation, and disassembly of protein complexes and degradation of misfolded proteins. The gene dnaJ which codes for Hsp40 in bacteria, modulate the activities of DnaK by acting as co-chaperone. In the present study, we cloned and expressed DnaK from Bacillus halodurans Guj1 were identified. The dnaK gene of B. halodurans was successfully expressed in E. coli BL21 (DE3) using pET-28a⁺ expression system. The open reading frame of the cloned gene contained 1839bp and encoded 612 amino acid residues. Calculated molecular weight and pI of the protein were 66.18kDa and 4.55 respectively. The deduced amino acid sequence of B. halodurans Guj1 showed about 60% identity with the E. coli counterpart. The 3D structure of dnaK from B. halodurans was constructed using the crystal structure of human HSP70 chaperone BiP as the template, which showed an identity of 88.8% together. Partially purified recombinant DnaK by heat treatment showed a band at approximately 70kDa on SDS-PAGE. Our findings showed that the recombinant DnaK improved the refolding efficiency of the carbonic anhydrase by 27% after 60min at 54°C. According to the results obtained, DnaK from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.